This invention relates to the field of control particles products for flow cytometry, and more particularly relates to a method for providing checks on pipetting precision and instrument linearity. The invention is particularly useful in a method for the absolute counting of cells, such as reticulocytes and leukocytes and more particularly one or more subsets of leukocytes, in a cell sample.
Flow cytometry comprises a well known methodology for identifying and distinguishing between different cell types in a non-homogeneous sample of cells. The sample may be drawn from a variety of sources such as blood, lymph, urine, or may be derived from suspensions of cells from solid tissues such as brain, kidney or liver. In the flow cytometer, cells are passed substantially one at a time through one or more sensing regions wherein each cell is illuminated by an energy source. The energy source generally comprises means that emits light of a single wavelength such as that provided by a laser (e.g., He/Ne or argon) or a mercury arc lamp with appropriate bandpass filters. Different sensing regions can include energy sources that emit light at different wavelengths.
In series with each sensing region, various light collection means, such as photomultiplier tubes, are used to gather light that is refracted by each cell (generally referred to as forward light scatter), light that is reflected orthogonal to the direction of the flow of the cells through a sensing region (generally referred to as orthogonal light scatter) and one or more light collection means to collect fluorescent light that may be emitted from the cell as it passes through a sensing region and is illuminated by the energy source. Light scatter is generally correlated with the physical characteristics of each cell.
Flow cytometers further comprise data recording and storage means, such as a computer, wherein separate channels record and store the light scatter and fluorescence data from each cell as it passes through a sensing region (i.e., all of the data collected for each cell comprise a xe2x80x9crecorded eventxe2x80x9d). By plotting orthogonal light scatter versus forward light scatter in either real time or by reanalysis of the data after the events have been recorded, one can distinguish between and count, for example, the granulocytes, monocytes and lymphocytes in a population of leukocytes. By gating on only lymphocytes, for example, using light scatter and by the use of appropriate cell markers, such as monoclonal antibodies labelled with fluorochromes of different emission wavelength and/or nucleic acid dyes, one can further distinguish between and count cell types within the lymphocyte population (e.g., between CD4+and CD8+lymphocytes). U.S. Pat. Nos. 4,727,020, 4,704,891 and 4,599,307 describe the arrangement of the various components that comprise a flow cytometer and also the general principles of its use.
Because the accurate measurement of light either scattered by or emitted from a cell is critical to the operation of a flow cytometer, assuring that the instrument is properly setup is important to its daily operation and use. There are a number of products available to xe2x80x9csetupxe2x80x9d a flow cytometer. These products generally comprise polymeric microparticles which are labelled with one or more fluorescent dyes and which come in one or more sizes. The instrument operator has the ability once or more a day to xe2x80x9ccalibratexe2x80x9d or xe2x80x9calignxe2x80x9d the instrument using these particles to be sure that the instrument is functioning properly. U.S. Pat. Nos. 5,073,497, 5,073,498 and 5,084,394 describe such beads and methods of use in setting up a flow cytometer. Co-pending and commonly assigned U.S. Ser. No. 897,616 (filed Jun. 10, 1992) further describes the use of cells as control particles for staining and instrument calibration.
Apart from issues involving the use and operation of the instrument, there are other factors which can effect results gathered by means of flow cytometry. While it is possible using the above-described methods to count the number of cells in a sample and to distinguish between various cell populations, the number of cells counted will be relative (i.e., it will not give an absolute count for a specific volume of blood, for example). Generally, these methods require that red blood cells be substantially removed from the sample. One reason is because the light scatter of the red blood cells and leukocytes is substantially overlapping making their differentiation based on light scatter alone difficult. Another reason is that in order to count leukocytes in a more rapid manner the number of red blood cells must be reduced because the number of red blood cells to leukocytes is approximately 1,000 to 1. Accordingly, practitioners in the field routinely lyse whole blood or separate out the blood cell components by density dependent centrifugation.
In addition to the step required for whole blood separation, other steps are routinely involved. For example, before or after lyses cell markers generally are added. Unbound markers, then, are routinely washed from the cells. After that step, a fixative is added. Finally, cells in solution are run on a flow cytometer. In some flow cytometers, all of the solution containing cells is delivered to the cytometer for analysis. In other flow cytometers, only a measured amount of solution is delivered.
While lysis and washing steps are routine, there are now methods and procedures that do not involve washing. These so-called xe2x80x9cno-washxe2x80x9d methods involve the addition of immunofluorescence markers and fixatives without washing steps. Co-pending and commonly assigned U.S. Ser. No. 846,316 (filed Mar. 5, 1992) is directed to one such method.
Regardless of the method or instrument used, any step in which the sample of cells is physically manipulated introduces not only the possibility for error, but also the potential for loss of cells from the sample. In addition, each step increases the risk to the technician of being exposed to contaminated blood.
Thus, in each of the presently described systems, there are one or more obstacles that limit ease in assuring instrument accuracy. These obstacles are not overcome by the mere addition of a reference particle, as described in U.S. Pat. No. 4,110,604, with flow cytometry or the occasional calibration of the instrument with control particles. Several drawbacks remain.
A major drawback to the use of flow cytometers is that unless the fluorescence channels and optical alignment of each flow cytometer is calibrated to read the same, there is no assurance as to the source of variation in a sample. It is likely that one instrument will give different readings on the same sample on different days if it was aligned and/or calibrated differently each day. Similarly, there is no assurance that any two instruments will provide the same results even if properly set up Accordingly, while flow cytometry provides a better measure of identifying and distinguishing between cells in a sample, its present use as a clinical instrument may be diminished by the limitations in set up and operation if not properly performed. What is required is a single system or method that will allow one to accurately count cells in a sample and/or be assured that the results from one instrument are consistent from sample to sample as well as consistent with results obtained from other instruments.
Even through the resolution of some of these problems through the methods and kits described in co-pending and commonly assigned U.S. Ser. No. 570,569 (filed Aug. 7, 1990), additional issues remain. For example, the use of a single reference particle will provide a means to perform absolute counts in a cell sample. The addition of such particles, however, does not assure that the technician handling the samples or the equipment used to handle the samples are accurate. Sample error introduced through the pipetting step can be a source of significant variation which cannot be otherwise accounted for. Similarly, the use of a single reference particle may not assure that the flow cytometer is accurately counting the particles, and thus, the cells. If the particle count is not accurate, the cell count cannot be accurate.
The present invention overcomes all of these obstacles and provides a one step test for absolute counting of one or more specific populations of cells in an unlysed whole blood sample. The present invention further provides a check on pipetting accuracy and provides a check on instrument linearity.
This invention, therefore, relates to a method and kit comprising control reagents for conventional (i.e., non-absolute) cell counting. In this method, a test sample is split into more than one aliquot, preferably two, more preferably four, and each aliquot is added to a separate tube. Each tube may contain one or more cell markers or the markers may be added as a separate step. To each tube is added a known concentration of microparticles. The concentration of microparticles in each tube will differ. Each tube then is analyzed by means of flow cytometry and the number of microparticles and cells per tube is counted. The result from counting should be linear if instrument linearity is correct. In the instance where two or more cell markers are being used to identify two or more subsets of cells, it is preferable to use at least four tubes and to add one cell marker to two tubes and the other cell marker to the two other tubes. In this instance, if the instrument is functioning properly, the coefficients of variation between tubes containing the same marker should be minimal.
The invention has particular utility in a method and kit for the absolute counting of one or more populations of cells in a sample. The preferred means for counting such cells comprises a flow cytometer. In this embodiment of the method, a test sample is added to a tube. The tube may contain a diluent. The diluent may comprise a mixture of a fixative, one or more cell markers and a known amount of a first microparticle. The first microparticle is fluorescent and the fluorescence is distinguishable from the fluorescence emitted by the cell marker(s). To this mixture, a known amount of a second fluorescent microparticle is added. The fluorescence of the second microparticle is distinguishable from the fluorescence emitted by the cell marker(s) and the first microparticle. The sample then is vortexed, incubated, vortexed again and run on a flow cytometer having one or more fluorescence channels.
In an alternative embodiment, the diluent may comprise only the cell markers. In that instance, the fixative and first microparticles are added separately. In another embodiment, the diluent may comprise the cell markers and first microparticles. The fixative is added separately. The timing of the addition of these various components is not critical to the practice of the invention.
Fluorescence data is recorded and stored for each event. A fluorescence trigger is set for one fluorescence channel so as to include essentially all of the microparticles and cells to be counted. The number of microparticles then is counted by analyzing the recorded events.
Counting the number of cells in the sample, the number of first microparticles and by knowing the amount of first microparticles added per unit volume, the number of cells in each population can be absolutely counted. Counting the number of second microparticles and the number of first microparticles in the sample permits a check on pipetting accuracy and instrument linearity.
A kit useful in the practice of this invention comprises the following items: a sample tube and a diluent wherein the diluent comprises a mixture of one or more cell markers and a known amount of a first microparticle. The kit also will contain two or more containers having different concentrations of a second microparticle. The diluent may be packaged in the tube. In the tube, the diluent may be liquid or may be dried by methods known to those skilled in the art such as lyophilization. Drying may be performed in the presence of a stabilization agent such as trehalose. In the dried format, the xe2x80x9cdiluentxe2x80x9d will be return to solution upon addition of the liquid sample such as blood. In any embodiment, the diluent may be separately contained or may be broken up into its several components each of which may be separately contained. In these alternatives, the diluent may be added to the sample tube before or after the sample is added to the tube.
Another kit useful in the practice of this invention comprises two or more tubes and two or more containers each container having a different amount of a fluorescent microparticle contained therein.